Ligand

ABSTRACT

The present invention provides a variant proliferation-inducing ligand (APRIL), which has a higher binding affinity to BCMA than wild-type APRIL; and/or altered binding kinetics compared with wild-type APRIL, and/or a higher BCMA:TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) binding ratio than wild-type APRIL and which comprises mutations at one or more of the following positions: A125, V174, T175, M200, P201, S202, H203, D205 and R206.

FIELD OF THE INVENTION

The present invention relates to a variant proliferation-inducing ligand(APRIL) which binds the B cell maturation antigen (BCMA). Therapeuticagents comprising such a variant APRIL are useful in the treatment ofplasma cell diseases such as multiple myeloma.

BACKGROUND TO THE INVENTION Multiple Myeloma

Multiple Myeloma (myeloma) is a bone-marrow malignancy of plasma cells.Collections of abnormal plasma cells accumulate in the bone marrow,where they interfere with the production of normal blood cells. Myelomais the second most common hematological malignancy in the U.S. (afternon-Hodgkin lymphoma), and constitutes 13% of haematologic malignanciesand 1% of all cancers. The disease is burdensome in terms of sufferingas well as medical expenditure since it causes pathological fractures,susceptibility to infection, renal and then bone-marrow failure beforedeath.

Unlike many lymphomas, myeloma is currently incurable. Standardchemotherapy agents used in lymphoma are largely ineffective formyeloma. In addition, since CD20 expression is lost in plasma cells,Rituximab cannot be used against this disease. New agents such asBortezamib and Lenolidomide are partially effective, but fail to lead tolong-lasting remissions.

There is thus a need for alternative agents for the treatment of myelomawhich have increased efficacy and improved long-term effects.

BCMA

BCMA, also known as TNFRSF17, is a plasma cell specific surface antigenwhich is expressed exclusively on B-lineage haemopoietic cells ordendritic cells. It is a member of the TNF receptor family. BCMA is notexpressed on naïve B cells but is up-regulated during B-celldifferentiation into plasmablasts, and is brightly expressed on memory Bcells, plasmablasts and bone marrow plasma cells. BCMA is also expressedon the majority of primary myeloma cells. Apart from low levels of mRNAdetected on dendritic cells, BCMA expression appears to be absent onother tissues, indicating the potential as a target for noveltherapeutics for multiple myeloma.

BCMA functions within a network of interconnected ligands and receptorswhich is shown schematically in FIG. 1. Two other TNF receptors sharethe ligands APRIL and BAFF with BCMA—transmembrane activator and calciummodulator and cyclophilin ligand interactor (TACI, also known asTNFRSF13B), which is found on activated T-cells and all B-cells; andBAFF-R (TNFRSF13C) which is predominantly expressed on B-lymphocytes.Multiple myeloma cells express TACI in some cases and BCMA in mostcases, but never BAFF-R.

The natural ligand APRIL is potentially useful as or as part of aBCMA-targeting therapeutic. However, cross-reaction with TACI ispotentially a problem, because TACI is found on activated T-cells andall B-cells, so treatment with an agent directed to BCMA on myelomacells may also cause a pathological depletion of non-cancerous B and Tcell subsets.

APRIL is also potentially useful in diagnostic applications to identifyplasma cells, in particular the presence of malignant plasma cells inconditions such as multiple myeloma. However, again, the capacity ofAPRIL to also bind TACI means that APRII-based diagnostics will alsoidentify generally activated T-cells and all B-cells, meaning that theresults are ambiguous.

There is thus a need to develop anti-BCMA therapeutics and diagnosticswhich are not associated with these disadvantages.

DESCRIPTION OF THE FIGURES

FIG. 1—Ligand Specificity and Function Assignment of APRIL and BAFF

B-cell-activating factor (BAFF, TNFSF13B) interacts with BAFF-Receptor(BAFF-R, TNFRSF13C), B-cell membrane antigen (BCMA, TNFRSF17) andtransmembrane activator and calcium modulator and cyclophilin ligandinteractor (TACI, TNFRSF13B) while A proliferation-inducing ligand(APRIL, TNFSF13) interacts with BCMA, TACI and proteoglycans. BAFF-Ractivation affects peripheral B-cell survival, while BCMA may affectplasma cell survival. APRIL interaction with proteoglycans involvesacidic sulphated glycol-saminoglycan side-chain containingamino-terminus of APRIL.

FIG. 2—Expression data of BCMA on Myeloma

Myeloma cells from bone marrow samples from 39 multiple myeloma patientswere isolated by a CD138+ magnetic bead selection. These cells werestained with the anti-BCMA monoclonal antibody J6MO conjugated with PE(GSK). Antigen copy number was quantified using PE Quantibrite beads(Becton Dickenson) as per the manufacturer's instructions. A box andwhiskers plot of antigen copy number is presented along with the range,interquartile and median values plotted. We found the range is348.7-4268.4 BCMA copies per cell with a mean of 1181 and a median of1084.9.

FIG. 3—Standard design of a Chimeric Antigen Receptor

The typical format of a chimeric antigen receptor is shown. These aretype I transmembrane proteins. An ectodomain recognizes antigen. This iscomposed of an antibody derived single-chain variable fragment (scFv)which is attached to a spacer domain. This in turn is connected to atransmembrane domain which acts to anchor the molecule in the membrane.Finally, this is connected to an endodomain which acts to transmitsintracellular signals to the cell. This is composed of one or moresignalling domains.

FIG. 4—Design of the different APRIL-based CARs generated.

The CAR design as shown in FIG. 3 was modified so that the scFv wasreplaced with a modified form of APRIL to act as an antigen bindingdomain: APRIL was truncated so that the proteoglycan bindingamino-terminus is absent. A signal peptide was then attached totruncated APRIL amino-terminus to direct the protein to the cellsurface. Three CARs were generated with this APRIL based binding domain:A. In the first CAR, the human CD8 stalk domain was used as a spacerdomain. B. In the second CAR, the hinge from IgG1 was used as a spacerdomain. C. In the third CAR, the hinge, CH2 and CH3 domains of humanIgG1 modified with the pva/a mutations described by Hombach et al (2010Gene Ther. 17:1206-1213) to reduce Fc Receptor binding was used as aspacer (henceforth referred as Fc-pvaa). In all CARs, these spacers wereconnected to the CD28 transmembrane domain and then to a tripartiteendodomain containing a fusion of the CD28, OX40 and the CD3-Zetaendodomain (Pule et al, Molecular therapy, 2005: Volume 12; Issue 5;Pages 933-41).

FIGS. 5A-5C—Annotated Amino acid sequence of the above three APRIL-CARS

FIG. 5A: Shows the annotated amino acid sequence of the CD8 stalk APRILCAR (SEQ ID NO: 76); FIG. 5B: Shows the annotated amino acid sequence ofthe APRIL IgG1 hinge based CAR (SEQ ID NO: 77); FIG. 5C: Shows theannotated amino acid sequence of the APRIL Fc-pvaa based CAR (SEQ ID NO:75).

FIGS. 6A-6C—Expression and ligand binding of different APRIL based CARsFIG. 6A. The receptors were co-expressed with a marker gene truncatedCD34 in a retroviral gene vector. Expression of the marker gene ontransduced cells allows confirmation of transduction. FIG. 6B. T-cellswere transduced with APRIL based CARs with either the CD8 stalk spacer,IgG1 hinge or Fc spacer. To test whether these receptors could be stablyexpressed on the cell surface, T-cells were then stained withanti-APRIL-biotin/Streptavidin APC and anti-CD34. Flow-cytometricanalysis was performed. APRIL was equally detected on the cell surfacein the three CARs suggesting they are equally stably expressed. FIG. 6C.Next, the capacity of the CARs to recognize TACI and BCMA wasdetermined. The transduced T-cells were stained with either recombinantBCMA or TACI fused to mouse IgG2a Fc fusion along with an anti-mousesecondary and anti-CD34. All three receptor formats showed binding toboth BCMA and TACI. A surprising finding was that binding to BCMA seemedgreater than to TACI. A further surprising finding was that although allthree CARs were equally expressed, the CD8 stalk and IgG1 hinge CARsappeared better at recognizing BCMA and TACI than that with the Fcspacer.

FIGS. 7A-7C—Function of the different CAR constructs.

Functional assays were performed of the three different APRIL basedCARs. Normal donor peripheral blood T-cells either non-transduced (NT),or transduced to express the different CARs. Transduction was performedusing equal titer supernatant. These T-cells were then CD56 depleted toremove non-specific NK activity and used as effectors. SupT1 cellseither non-transduced (NT), or transduced to express BCMA or TACI wereused as targets. Data shown is mean and standard deviation from 5independent experiments. FIG. 7A. Specific killing of BCMA and TACIexpressing T-cells was determined using Chromium release. FIG. 7B.Interferon-γ release was also determined. Targets and effectors wereco-cultured at a ratio of 1:1. After 24 hours, Interferon-γ in thesupernatant was assayed by ELISA. FIG. 7C. Proliferation/survival of CART-cells were also determined by counting number of CAR T-cells in thesame co-culture incubated for a further 6 days. All 3 CARs directresponses against BCMA and TACI expressing targets. The responses toBCMA were greater than for TACI.

FIG. 8—Killing of primary Myeloma cells by APRIL CAR T-cells

Since most primary myeloma cells express a low number of BCMA moleculeson their surface, it was investigated whether killing of primary myelomacells occurs despite low-density expression. Three cases were selectedwhich represented the range of BCMA expression described in FIG. 2: thefirst had dim expression (lower than mean); the second case hadintermediate expression (approximately mean expression) and the thirdhad bright (above mean expression). A histogram of BCMA staining againstisotype control for all three cases is shown on the left. In this assay,only the CD8 stalk and hinge APRIL CARs were tested. On the left,survival of myeloma cells compared with starting numbers is shown at day3 and day 6 after a 1:1 co-culture of myeloma cells and CAR T-cells. Byday 6, >95% of the myeloma cells were eliminated, including those withdim BCMA expression.

FIGS. 9A-9C—Methods used to develop novel APRIL mutants useful for BCMAtargeting.

FIG. 9A. Candidate APRIL molecules were displayed in the CD8 stalk CARformat (but without a signalling endodomain) and were co-expressed withCD34 using a foot-and-mouth disease 2A sequence. FIG. 9B. Residues whichappeared important for BCMA specificity or affinity fromcrystallographic data were randomized by splicing-by-overlap PCR usingoligonucleotides which were degenerate over the coding codon as primers.These PCR products were ligated into the CD8 stalk CAR format and usedto transform bacteria. Individual bacterial colonies (each containing asingle mutant) were cultured. Plasmid DNA was isolated from thesecultures and used to transfect 293T cells. After transfection, 293Tcells were stained with either BCMA-Fc fusion or TACI-Fc fusionseparately, along with the marker gene. FIG. 9C. How relative binding toBCMA and TACI was estimated during screening: the slope of fluorescentintensity of CD34 staining versus either BCMA or TACI was calculated.Next, the ratio of this slope to that of wild-type APRIL was calculated.This value was used as the read-out.

FIG. 10—Summary of APRIL mutants—single residue mutations.

Mutations which show altered binding to BCMA-Fc and TACI-Fc aresummarized in comparison with that of wild type APRIL.

FIG. 11—Summary of APRIL mutants—multiple residue mutations.

Promising mutants were crossed either once or multiply with othermutants and characterized. Altered binding to BCMA-Fc and TACI-Fc isshown here again compared to wild-type APRIL.

FIGS. 12A-12F—BCMA-FC, TACI-FC and APRIL binding of some selectedmutants

Flow cytometry plots of selected mutants are shown in a table. The firstcolumn shows BCMA-Fc staining vs that of CD34. The second column showsTACI-Fc staining vs that of CD34. The third column shows APRIL stainingvs that of CD34. The first row shows wild-type APRIL staining as acontrol. The second row shows CD34 alone control.

FIG. 13—Vector co-expressing APRIL based CAR with truncated CD34

A cell line expressing the vector used for screening was incubated witheither BCMA-Fc or TACI-Fc and stained with both anti-CD34 andanti-human-Fc PE and FITC conjugated mAbs. The cells were then studiedby flow-cytometery. This shows a typical pattern of binding of BCMA andTACI relative to the marker gene CD34.

FIG. 14A—Schematic diagram illustrating a classical CAR

FIG. 14B: Design of the different APRIL-based CARs generated.

A signal peptide ias attached to truncated APRIL amino-terminus. Thiswas fused to different spacers: either the hinge, CH2 and CH3 domains ofhuman IgG1 modified with the pvaa mutation described by Hombach et al(2010 Gene Ther. 17:1206-1213) to reduce Fc Receptor binding; the stalkof human CD8α; and the hinge of IgG1. These spacers were connected to atripartite endodomain containing CD28 transmembrane domain, the OX40endodomain and the CD3-Zeta endodomain.

FIG. 15—Expression of different CARs

The receptors were co-expressed with enhanced blue fluorescence protein2 (eBFP2) using an IRES sequence. Primary human T-cells were transducedand stained with anti-APRIL-biotin/Streptavidin APC. Flow-cytometricanalysis was performed. eBFP2 signal is shown against APRIL detection.All three CARs are stably expressed (representative experiment of 3independent experiments performed using 3 different normal donorT-cells).

FIG. 16—Chromium release assay

Using normal donor peripheral blood T-cells either non-transduced (NT),or transduced to express different spacer CARs as effectors, and SupT1cells either non-transduced (NT), or transduced to express BCMA or TACIas targets. The T-cells were CD56 depleted to reduce NK activity. Thisis a representative of three independent experiments and is shown as anexample. Cumulative killing data is shown in FIG. 7A. Specific killingof BCMA and TACI expressing T-cells is seen with no activity againstnegative target cells.

FIG. 17—Interferon-gamma release

From a 1:1 co-culture of effectors and targets is measured by ELISA. TheCD8 stalk construct appears to have the best specificity while the hingeconstruct results in the most Interferon release demonstrates somenon-specific activity. This is representative of 3 independentexperiments and is shown as an example. Cumulative interferon-gammarelease data is shown in FIG. 7B.

FIG. 18—Examples of BCMA expression on primary myelomas

Four examples of myeloma samples stained with the rat anti-human BCMAmAb Vicky1 is shown. The first panel shows bright BCMA staining in apatient with a plasma cell leukemia (an unusual, advanced and aggressiveform of myeloma). The other three cases are clinically andmorphologically typical myelomas. They show the intermediate or dimstaining typically seen. Staining with isotype control (grey) issuperimposed. These are examples of cumulative BCMA expression datashown in FIG. 2.

FIGS. 19A-19C—Amino acid sequence of APRIL-CARS with a V5 epitope tag.

FIG. 19A: dAPRIL-HCH2CH3pvaa-CD28OXZ (SEQ ID NO: 78)

FIG. 19B: dAPRIL-CD8STK-CD28OXZ (SEQ ID NO: 79)

FIG. 19C: dAPRIL-HNG-CD28OXZ (SEQ ID NO: 80)

Sequences in this figure differ from those in FIG. 5 have a differentsignal peptide and no V5 tag.

FIGS. 20A-20W—Summary of screening BCMA specific APRIL mutants

Altered binding to BCMA-Fc and TACI-Fc. This is an example of initialscreening data miniprep DNA. Mutants were screened in batches withinter-experimental variation corrected for by expressed the average MFIgradient of APRIL mutant compared to wild type APRIL checked with eachbatch.

FIGS. 21A-21B—Sequence alignment of BCMA specific APRIL mutants

Minipreps selected during the random mutagenesis process were screenedfor expression by staining with BCMA-Fc and TACI-Fc. Mutants withpotentially useful or informative phenotypes were sequenced bycapilliary sequences and aligned with the original APRIL sequence theywere derived from. Shown in this figure are alignments of examplemutants identified during such a screening process. M200X—SEQ ID NO:102; M200C—SEQ ID NO: 103; M200L—SEQ ID NO: 104; M200S—SEQ ID NO: 105;M200*—SEQ ID NO: 106; M200A—SEQ ID NO: 107; M200G—SEQ ID NO: 108;M200N—SEQ ID NO: 109; P201X—SEQ ID NO: 110; P201G-4_406.seq—SEQ ID NO:111; P201A-18_406.seq—SEQ ID NO: 112; P201V-38_406.seq—SEQ ID NO: 113;P201A-46_406.seq—SEQ ID NO: 114; P201A-28_406.seq—SEQ ID NO: 115;P201A-44_406.seq—SEQ ID NO: 116; S202X—SEQ ID NO: 117; S202G-5_406—SEQID NO: 118; S202P-20_406—SEQ ID NO: 119; S202F-22_406.seq—SEQ ID NO:120; S202V_H203N-26_4—SEQ ID NO: 121; S202D-40_406.seq—SEQ ID NO: 122;T175X—SEQ ID NO: 123; T175G_S202G-4_40—SEQ ID NO: 124;T175G_S202V-6_40—SEQ ID NO: 125; T175H-11_406.seq—SEQ ID NO: 126;T175P-15_406.seq—SEQ ID NO: 127; T175H-16_406.seq—SEQ ID NO: 128;T175G-19_406.seq—SEQ ID NO: 129; T175A_S202E-24_4—SEQ ID NO: 130;T175S_S202G-25_4—SEQ ID NO: 131; V174X—SEQ ID NO: 132;V174T_S202V-1_40—SEQ ID NO: 133; V174G-4_406.seq—SEQ ID NO: 134;V174G_S202E-7_40—SEQ ID NO: 135; V174G_S202A-10_4—SEQ ID NO: 136;V174G_S202G-15_4—SEQ ID NO: 137; V174H_S202G-31_4—SEQ ID NO: 138;V174E_S202Y-41_4—SEQ ID NO: 139; D205X R206X—SEQ ID NO: 140;D205P-1_406.seq—SEQ ID NO: 141; D205R-R206G-27_4—SEQ ID NO: 142;D205P-R206K-33_4—SEQ ID NO: 143; D205P-R206N-35_4—SEQ ID NO: 144;D205P-R2061-44_4—SEQ ID NO: 145; D205S-R206H-4_40—SEQ ID NO: 146;D205Y-R206stop-1—SEQ ID NO: 147; D205+C-12_406—SEQ ID NO: 148;D205H-R206L-16_4—SEQ ID NO: 149; D205S-R206P-22_4—SEQ ID NO: 150;A125X—SEQ ID NO: 151; A1251-5_406—SEQ ID NO: 152.

FIG. 22—Graph of altered BCMA and TACI binding with glycinesubstitutions at targeted residues

FIGS. 23A-23B—Demonstration of in vivo function of APRIL CAR T-cells

Six 3 month old female NSG mice received 1×10⁷ MM1.s.FLuc cells vialtail-vein injection. Mice were imaged with bioluminescence at day 8 andday 13. After imaging on day 13, four mice received 5×10⁶ APRIL CART-cells via tail vein injection. Mice were imaged on day 13 and day 18.Mice which received CAR T-cells are indicated with (*). Remission ofMyeloma could be observed by Day 18 in all treated mice, while diseasein untreated mice progressed.

FIGS. 24A-24G: Different APRIL-BiTE formats designed and constructed

FIG. 24A: OKT3 scFv connected to truncated APRIL by the IgG1 hinge; FIG.24B: OKT3 scFv connected to truncated APRIL via a (SGGGGS)3 linker; FIG.24C: OKT3 scFv connected to truncated APRIL via the CD8 stalk; FIG. 24D:truncated APRIL connected to OKT3 scFv via an IgG1 hinge; FIG. 24E:truncated APRIL connected to the OKT3 scFv via a (SGGGGS)3 linker; FIG.24F truncated APRIL connected to the OKT3 scFv via a CD8 spacer.Constructs in FIG. 24C and FIG. 24F should form homodimers throughdisulphide bonds in the CD8 spacer.

FIG. 24G: schematic diagram of molecular clustering on the cell-to-cellinterface upon binding of the APRILiTE.

FIG. 25—Western blot of supernatant from 293T cells transfected with thedifferent APRILiTE constructs. Blotting was done with anti-APRIL.

FIG. 26A—Binding of APRILiTES 1, 3 and 6 to wild-type SupT1 cells andSupT1 cells engineered to express BCMA and TACI. Staining is withanti-APRIL biotin/Streptavidin APC. Aprilites show no binding to WTSupT1 cells but bind to BCMA expressing cells, and to a lesser extent toTACI expressing cells.

FIG. 26B—Binding of APRILiTEs to wild-type Jurkats, but not to Jurkatswith no T-cell receptor. This demonstrates that the APRILiTES bind theT-cell receptor.

FIG. 27—Co-culture of T-cells 1:1 non-transduced or engineered SupT1cells in the presence of blank media or the 3 APRILiTES.

FIG. 28—Complete deletion of BCMA expressing SupT1 cells was observedafter 3 day co-culture in the presence of APRILiTE 1,3 and 6.

FIG. 29—Examples of BCMA expression on primary myelomas. Four examplesof myeloma samples stained with the rat anti-human BCMA mAb Vicky1 isshown. The first panel shows bright BCMA staining in a patient with aplasma cell leukemia (an unusual, advanced and aggressive form ofmyeloma). The other three cases are clinically and morphologicallytypical myelomas. They show the intermediate or dim staining typicallyseen. Staining with isotype control (grey) is superimposed.

FIGS. 30A-30C—Amino acid sequence of APRILiTEs

FIG. 30A: APRILiTE#01 (SEQ ID NO: 96); FIG. 30B: APRILiTE#03 (SEQ ID NO:97); FIG. 30C: APRILiTE#06 (SEQ ID NO: 98)

FIG. 31—Staining of myeloma samples for BCMA overlaid on isotypecontrol. These myeloma cells express BCMA but at low levels

FIG. 32—Low-power microscopy of co-cultures and controls at day 1. Clearclumping/activation of T-cells can be seen when cultured with myelomacells in the presence of an APRILiTE.

FIG. 33—Inteferon-gamma release with myeloma cells alone, co-culturedwith peripheral blood T-cells, both together in the absence of andpresence of APRILiTES#3 and #6

FIG. 34—Survival at day 6 of co-culture of myeloma cells in culture.Both APRILiTES tested result in efficient killing of primary myelomacells in the presence of PBMCs.

FIG. 35—Testing function of various APRIL mutants in a BITE format

Four normal donor PBMCs were incubated with SupT1 cells, SupT1 cellsengineered to express BCMA, SupT1 cells engineered to express TACI oralone in the presence of different BiTES based on either WT APRIL orvarious mutants. Interferon-gamma levels were measured 24 hours later.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have activated developed mutants of theBCMA-binding ligand APRIL, which have a higher BCMA:TACI binding ratiothat wild-type APRIL. These mutants exhibit a greater degree ofspecificity for BCMA so provide more focussed targeting ofBCMA-expressing cells for therapeutic and diagnostic applications.

Thus, in a first aspect the present invention provides a variantproliferation-inducing ligand (APRIL), which has a higher bindingaffinity to BCMA than wild-type APRIL; and/or altered binding kineticscompared with wild-type APRIL, and/or a higher BCMA:TACI (transmembraneactivator and calcium modulator and cyclophilin ligand interactor)binding ratio than wild-type APRIL and which comprises mutations at oneor more of the following positions: A125, V174, T175, M200, P201, S202,H203, D205 and R206.

The variant APRIL may comprise one of following the single mutations:

A125T, V174T, V174G, T175H, T175S, T175G, M200C, M200L, M200G, M200S,M200A, M200N, P201V, P201A, P201G, P201R, P201Y, P201W, S202G, S202F,S202D, S202V, S202P, D205P.

The variant APRIL may comprise a combination of mutations at thefollowing positions: V174 and T175; or V174 and M200; or V174 and S202;or V175 and M200, or V175 and S202; or D205 and R206; or V174, T175 andM200; or V174, T175 and S202; or T175, D205 and R206; or M200, D205 andR206; or V174, T175, M200 and S202; or T175, S202, D205 and R206.

The variant APRIL may comprise one of the following mutationcombinations:

V174T and T175A; or V174T and M200G; or T174S and S202G; or V174T andS202V; or V174G and S202G, or V174G and S202E; or V174G and S202A; orV174G and S202G; or V174E and S202Y; or T175A and S202E; or T175G andS202G; or T175G and S202V; or T175A and S202P; or T175A and M200G; orT175S and S202G; or S202V and H203N; or D205H and R206L; or D205P andR206K; or D205P and R206N; or D205S and R206P; or D205R and R206G; orD205P and R2061; or D205S and R206H; or V174T, T175A and S202E; orV174T, T175A and M200G; or T175A, D205P and R206N; or T175A, D205S andR206H; or M200G, D205P and R206N; or M200G, D205S and R206H; or V174T,T175A, M200G and S202E; or T175A, S202E, D205P and R206N; or T175A,S202E, D205S and R206H.

The present invention also provides a variant proliferation-inducingligand (APRIL) which comprises the mutation M200G.

The present invention also provides a chimeric antigen receptor (CAR)which comprises an antigen-binding domain, a transmembrane domain and anendodomain, wherein the antigen-binding domain comprises a variant APRILaccording to any the first aspect of the invention.

The present invention also provides a bispecific T-cell engager (BiTE)which comprises and antigen-binding domain and a T-cell activationdomain, wherein the antigen-binding domain comprises a variant APRILaccording to the first aspect of the invention.

In a second aspect, the present invention provides a nucleic acidsequence encoding a variant APRIL according to the first aspect of theinvention, or a CAR or BiTE comprising such a variant APRIL.

In a third aspect the present invention provides a vector comprising anucleic acid sequence according to the second aspect of the invention.

The present invention also provides a cell which comprises a chimericantigen receptor comprising a variant APRIL according to the firstaspect of the invention.

The present invention also provides a method for making such a cellwhich comprises the step of transducing or transfecting a cell with avector according to the third aspect of the invention which comprises anucleic acid sequence encoding a chimeric antigen receptor.

In a fourth aspect, the present invention provides a therapeutic agentwhich comprises a variant APRIL according to the first aspect of theinvention, a CAR or BiTE comprising such a variant APRIL, a cellcomprising such a CAR, a nucleic acid according to the second aspect ofthe invention or a vector according to the third aspect of theinvention.

There is also provided a method for treating a plasma cell disorderwhich comprises the step of administering a therapeutic agent accordingto the fourth aspect of the invention to a subject.

There is also provided a therapeutic agent according to the fourthaspect of the invention for use in treating a plasma cell disorder.

There is also provided the use of a therapeutic agent according to thefourth aspect of the invention in the manufacture of a medicament fortreating a plasma cell disorder.

In a fifth aspect, the present invention provides a diagnostic agent fordetecting plasma cells which comprises a variant APRIL according to thefirst aspect of the invention.

There is also provided the diagnostic agent according to the fifthaspect of the invention for diagnosing a plasma cell disorder.

There is also provided a method for diagnosing a plasma cell disorder ina subject in vivo which comprises the step of administering a diagnosticagent according to the fifth aspect of the invention to the subject.

The is also provided a method for diagnosing a plasma cell disorder in asubject which comprises the step of adding a diagnostic agent accordingto the fifth aspect of the invention to a sample from the subject invitro.

The sample may be, or be derived from, a blood sample.

The plasma cell disorder may be selected from: plasmacytoma, plasma cellleukemia, multiple myeloma, macroglobulinemia, amyloidosis,Waldenstrom's macroglobulinemia, solitary bone plasmacytoma,extramedullary plasmacytoma, osteosclerotic myeloma, heavy chaindiseases, monoclonal gammopathy of undetermined significance andsmoldering multiple myeloma.

The plasma cell disorder may be multiple myeloma.

DETAILED DESCRIPTION April

The present invention relates to a variant proliferation-inducing ligand(APRIL), which has a higher binding affinity to BCMA than wild-typeAPRIL; and/or altered binding kinetics compared with wild-type APRIL,and/or a higher BCMA:TACI (transmembrane activator and calcium modulatorand cyclophilin ligand interactor) binding ratio than wild-type APRIL.APRIL is also known as TNFSF13.

The term “variant” is synonymous with “mutant” or “engineered” and meansAPRIL comprising one or more mutations, such as substitution(s),addition(s) or deletions(s). Typically the mutation is a substitution.

The wild-type sequence of APRIL is available at UNIPROT/075888 and isshown below (SEQ ID No. 1). It is not a classical secreted protein inthat it has no signal peptide. It has a furin cleavage site “KQKKQK”(underlined in SEQ ID No. 1). The amino terminus is involved inproteoglycan binding.

Kimberley et al (2009, FASEB J 23:1584-1595) is a study investigatingthe role of heparin sulphate proteoglycan (HSPG) interaction in APRILsignalling. Point mutations were generated as follows:

1) APRIL-triple (designated WT-triple), containing 3 point mutations:R146S, R189S, H220E;2) APRIL-HSPG (designated HSPG), containing three point mutations in thehydrophobic motif (QKQKK¹¹³Q);3) APRIL-HSPG-triple (designated HSPG-triple), in which all 6 aminoacids were mutated at both these sites.4) APRIL-R231A, a form of APRIL capable of binding HSPGs but lacking theability to bind either TACI or BCMA (FIG. 2) which comprises a keyarginine to alanine mutation within the receptor binding region.

All mutants except APRIL-R231A retained the ability to bind both BCMAand TACI. The R231A mutant showed complete loss of binding to bothreceptors but retained its ability to bind HSPGs.

The variant APRIL of the present invention may comprise the BCMA-bindingsite of APRIL. The variant APRIL may comprise a fragment of APRIL whichcomprises the BCMA-binding site.

The variant APRIL may comprise a truncated APRIL, which lacks the aminoterminal end of the molecule. The truncated APRIL may retain BCMA andTACI binding but lose proteoglycan binding. Truncated APRIL can becleaved at or immediately after the furin cleavage site. Truncated APRILmay lack the amino terminal 116 amino acids from the wild-type APRILmolecule shown as SEQ ID No. 1. Truncated APRIL may comprise thesequence shown as SEQ ID No. 2 (which corresponds to the portion of SEQID No. 1 shown in bold) or a variant thereof. This corresponds to theportion of the molecule which is needed for BCMA and TACI binding.

SEQ ID No. 1        10         20         30         40         50         60MPASSPFLLA PKGPPGNMGG PVREPALSVA LWLSWGAALG AVACAMALLT QQTELQSLRR        70         80         90        100        110        120EVSRLQGTGG PSQNGEGYPW QSLPEQSSDA LEAWENGERS RKRRAVLTQ K QKKQH SVLHL       130        140        150        160        170        180VPINATSKDD SDVTEVMWQP ALRRGRGLQA QGYGVRIQDA GVYLLYSQVL FQDVTFTMGQ       190        200        210        220        230        240VVSREGQGRQ ETLFRCIRSM PSHPDRAYNS CYSAGVFHLH QGDILSVIIP RARAKLNLSP       250 HGTFLGFVKL SEQ ID No. 2VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKL

The variant APRIL or variant truncated APRIL has binding characteristicswhich make it more specific that wild-type APRIL. For instance, in someembodiments or applications, the variant APRIL has higher affinity toBCMA than wild-type APRIL. In some embodiments or applications, thevariant APRIL has different binding kinetics to BCMA than wild-typeAPRIL. In some applications, the variant APRIL has a BCMA:TACI bindingratio is higher than wild-type APRIL or a combination thereof. Themutant APRIL comprises mutations at one or more of the followingpositions: A125, V174, T175, M200, P201, S202, H203, D205 and R206(shown in grey in SEQ ID No. 1).

In particular, the variant APRIL may comprise one of following thesingle mutations: (SEQ IDs 3 to 26):

-   -   A125T,    -   V174T, V174G,    -   T175H, T175S, T175G,    -   M200C, M200L, M200G, M200S, M200A, M200N,    -   P201V, P201A, P201G, P201R, P201Y, P201W,    -   S202G, S202F, S202D, S202V, S202P, D205P.

These mutations have been determined to alter binding to BCMA and TACIin a manner which may be useful to BCMA targeting. The relative bindingto BCMA and TACI is shown in Table 1, illustrated in FIG. 10 with someexamples shown in FIG. 12A-12F.

TABLE 1 Mutation % BCMA WT % TACI WT Sequence A125T  46  42 SEQ ID 13V174T 379 500 SEQ ID 14 V174G 109  34 SEQ ID 15 T175H 144  78 SEQ ID 16T175S 129  35 SEQ ID 17 T175G  67  41 SEQ ID 18 M200C  50   0 SEQ ID 19M200L 164  62 SEQ ID 20 M200G  35   0 SEQ ID 21 M2005  10   0 SEQ ID 22M200A  20   3 SEQ ID 23 M200N  12   4 SEQ ID 24 P201V  20   1 SEQ ID 25P201A  24  18 SEQ ID 26 P201G  13   4 SEQ ID 27 P201R   8   3 SEQ ID 28P201Y   9   0 SEQ ID 29 P201W   6   5 SEQ ID 30 S202G 116  68 SEQ ID 31S202F  28  30 SEQ ID 32 S202D  30  32 SEQ ID 33 S202V 204 232 SEQ ID 34S202P 163 218 SEQ ID 35 D205P  26  18 SEQ ID 36 SEQ ID 3 (A125T)VLHLVPINTTSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 4 (V174T)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDTTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 5 (V174G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDGTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 6 (1175H)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVHFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 7 (1175S)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVSFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 8 (1175G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVGFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 9 (M200C)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSCPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 10 (M200L)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSLPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 11 (M200G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSGPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 12 (M200S)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSSPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 13 (M200A)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSAPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 14 (M200N)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSNPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 15 (P201V)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMVSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 16 (P201A)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMASHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 17 (P201G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMGSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 18 (P201Y)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMYSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 19 (P201R)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMRSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 20 (P201W)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMWSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 21 (5202G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPGHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 22 (5202F)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPFHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 23 (5202D)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPDHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 24 (5202V)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPVHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 25 (5202P)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPPHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 26 (D205P)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPPRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKL

The variant APRIL may comprise a combination of mutations at thefollowing positions: V174 and T175; or V174 and M200; or V174 and S202;or V175 and M200, or V175 and S202; or S202 and H203; or D205 and R206;or V174, T175 and M200; or V174, T175 and S202; or T175, D205 and R206;or M200, D205 and R206; or V174, T175, M200 and S202; or T175, S202,D205 and R206;

In particular, the variant APRIL may comprise one of the followingspecific combined mutations:

-   -   V174T and T175A; or V174T and M200G; or T174S and S202G; or    -   V174T and S202V; or V174G and S202G, or V174G and S202E; or    -   V174G and S202A; or V174G and S202G; or V174E and S202Y; or    -   T175A and S202E; or T175G and S202G; or T175G and S202V; or    -   T175A and S202P; or T175A and M200G; or T175S and S202G; or    -   S202V and H203N; or D205H and R206L; or D205P and R206K; or    -   D205P and R206N; or D205S and R206P; or D205R and R206G; or    -   D205P and R2061; or D205S and R206H; or    -   V174T, T175A and S202E; or V174T, T175A and M200G; or    -   T175A, D205P and R206N; or T175A, D205S and R206H; or    -   M200G, D205P and R206N; or M200G, D205S and R206H; or    -   V174T, T175A, M200G and S202E; or    -   T175A, S202E, D205P and R206N; or    -   T175A, S202E, D205S and R206H.

These specific combined mutations have been shown to alter binding toBCMA and TACI in a manner which is useful to BCMA targeting (see Table 2and FIG. 11).

TABLE 2 Mutation % BCMA WT % TACI WT Sequence V174T, T175A 131  80SEQ ID 27 V174T, M200G 172  49 SEQ ID 28 T174S, S202G  43  13 SEQ ID 29V174T, S202V 303 613 SEQ ID 30 V174G, S202G  67  24 SEQ ID 31V174G, S202E  35  18 SEQ ID 32 V174G, S202A 132  36 SEQ ID 33V174G, S202G  29  49 SEQ ID 34 V174E, S202Y  33  15 SEQ ID 35T175A, S202E  87  15 SEQ ID 36 T175G, S202G  34  17 SEQ ID 37T175G, S202V  59  30 SEQ ID 38 T175A, S202P 100   0 SEQ ID 39T175A, M200G  14   1 SEQ ID 40 T175s, s202G  43  13 SEQ ID 415202V, H203N  11  24 SEQ ID 42 D205H, R206L 357  86 SEQ ID 43D205P, R206K 255  90 SEQ ID 44 D205P, R206N 111 138 SEQ ID 45D205s, R206P 420  81 SEQ ID 46 D205R, R206G 404  84 SEQ ID 47D205P, R2061 343  54 SEQ ID 48 D205s, R206H 234 112 SEQ ID 49V174T, T175A, S202E 186  87 SEQ ID 50 V174T, T175A, M200G  28   4SEQ ID 51 T175A, D205P, R206N  13   1 SEQ ID 52 T175A, D205s, R206H  15  2 SEQ ID 53 M200G, D205P, R206N  53   4 SEQ ID 54 M200G, D205S, T206H 68  15 SEQ ID 55 V174T, T175A, M200G, S202E  43   0 SEQ ID 56T175A, S202E, D205P, T206N  19   0 SEQ ID 57 T175A, S202E, D205S, T206H 28   0 SEQ ID 58 SEQ ID 27 (V174T, T175A)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDTAFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 28 (V174T, M200G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDTTFTMGQVVSREGQGRQETLFRCIRSGPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 29 (T174S, S202G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDSTFTMGQVVSREGQGRQETLFRCIRSMPGHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 30 (V174T, S202V)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDTTFTMGQVVSREGQGRQETLFRCIRSMPVHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 31 (V174G, S202G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDGTFTMGQVVSREGQGRQETLFRCIRSMPGHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 32 (V174G, S202E)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDGTFTMGQVVSREGQGRQETLFRCIRSMPEHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 33 (V174G, S202A)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDGTFTMGQVVSREGQGRQETLFRCIRSMPAHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 34 (V174G, S202G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDGTFTMGQVVSREGQGRQETLFRCIRSMPGHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 35 (V174E, S202Y)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDETFTMGQVVSREGQGRQETLFRCIRSMPYHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 36 (T175A, S202E)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVAFTMGQVVSREGQGRQETLFRCIRSMPEHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 37 (T175G, S202G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVGFTMGQVVSREGQGRQETLFRCIRSMPGHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 38 (T175G, S202V)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVGFTMGQVVSREGQGRQETLFRCIRSMPVHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 39 (T175A, S202P)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVAFTMGQVVSREGQGRQETLFRCIRSMPPHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 40 (T175A, M200G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVAFTMGQVVSREGQGRQETLFRCIRSGPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 41 T175S, S202GVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVSFTMGQVVSREGQGRQETLFRCIRSMPGHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 42 (S202V, H203N)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPVNPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 43 (D205H, R206L)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPHLAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 44 (D205P, R206K)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPPKAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 45 (D205P, R206N)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPPNAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 46 (D205S, R206P)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPSPAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 47 (D205R, R206G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPRGAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 48 (D205P, R206I)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPPIAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 49 (D205S, R206H)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPSHAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 50 (V174T, T175A, S202E)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDTAFTMGQVVSREGQGRQETLFRCIRSMPEHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 51 (V174T, T175A, M200G)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDTAFTMGQVVSREGQGRQETLFRCIRSGPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 52 (T175A, D205P, R206N)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVAFTMGQVVSREGQGRQETLFRCIRSMPSHPPNAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 53 (T175A, D205S, R206H)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVAFTMGQVVSREGQGRQETLFRCIRSMPSHPSHAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 54 (M200G, D205P, R206N)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSGPSHPPNAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 55 (M200G, D205S, R206H)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSGPSHPSHAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 56 (V174T, T175A, M200G, S202E)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDTAFTMGQVVSREGQGRQETLFRCIRSGPEHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 57 (T175A, S202E, D205P, R206N)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVAFTMGQVVSREGQGRQETLFRCIRSMPEHPPNAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKLSEQ ID 58 (T175A, S202E, D205S, R206H)VLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVAFTMGQVVSREGQGRQETLFRCIRSMPEHPDHAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHG TFLGFVKL

T Cell Activation

The present invention also provides a bi-specific molecule whichcomprises

-   -   (i) a first domain which binds B cell maturation antigen (BCMA)        and comprises a mutant APRIL according to the first aspect of        the invention; and    -   (ii) a second domain capable of activating a T-cell.

The second domain of the molecule of the present invention is capable ofactivating T cells. T cells have a T cell-receptor (TCR) at the cellsurface which recognises antigenic peptides when presented by an MHCmolecule on the surface of an antigen presenting cell. Such antigenrecognition results in the phosphorylation of immunoreceptortyrosine-based activation motifs (ITAMs) by Src family kinases,triggering recruitment of further kinases which results in T cellactivation including Ca²⁺ release.

The second domain may cause T cell activation by triggering the samepathway triggered by antigen-specific recognition by the TCR.

Cluster of Differentiation 3 (CD3)

The second domain of the bi-specific molecule of the invention may bindCD3.

CD3 is a protein complex composed of four distinct chains: a CD3γ chain,a CD3δ chain, and two CD3δ chains. CD3 associates with the T-cellreceptor (TCR) and the ζ-chain on the surface of a T cell to generate anactivation signal. The TCR, ζ-chain, and CD3 molecule together comprisethe TCR complex.

Clustering of CD3 on T cells, e.g. by immobilized anti-CD3-antibodies,leads to T cell activation similar to the engagement of the T cellreceptor, but independent from its clone typical specificity.

Due to its central role in modulating T cell activity, there have beenattempts to develop molecules that are capable of binding TCR/CD3. Muchof this work has focused on the generation of antibodies that arespecific for the human CD3 antigen.

The second domain may comprise an antibody or part thereof whichspecifically binds CD3, such as OKT3, WT32, anti-leu-4, UCHT-1, SPV-3TA,TR66, SPV-T3B or affinity tuned variants thereof.

As used herein, “antibody” means a polypeptide having an antigen bindingsite which comprises at least one complementarity determining regionCDR. The antibody may comprise 3 CDRs and have an antigen binding sitewhich is equivalent to that of a domain antibody (dAb). The antibody maycomprise 6 CDRs and have an antigen binding site which is equivalent tothat of a classical antibody molecule. The remainder of the polypeptidemay be any sequence which provides a suitable scaffold for the antigenbinding site and displays it in an appropriate manner for it to bind theantigen. The antibody may be a whole immunoglobulin molecule or a partthereof such as a Fab, F(ab)′₂, Fv, single chain Fv (ScFv) fragment,Nanobody or single chain variable domain (which may be a VH or VL chain,having 3 CDRs). The antibody may be a bifunctional antibody. Theantibody may be non-human, chimeric, humanised or fully human.

Alternatively the second domain may comprise a CD3-binding moleculewhich is not derived from or based on an immunoglobulin. A number of“antibody mimetic” designed repeat proteins (DRPs) have been developedto exploit the binding abilities of non-antibody polypeptides. Suchmolecules include ankyrin or leucine-rich repeat proteins e.g. DARPins(Designed Ankyrin Repeat Proteins), Anticalins, Avimers and Versabodies.

The second domain of the bi-specific molecule of the invention maycomprise all or part of the monoclonal antibody OKT3, which was thefirst monoclonal antibody approved by the FDA. OKT3 is available fromATCC CRL 8001. The antibody sequences are published in U.S. Pat. No.7,381,803.

The second domain may comprise one or more CDRs from OKT3. The secondbinding domain may comprise CDR3 from the heavy-chain of OKT3 and/orCDR3 from the light chain of OKT3. The second binding domain maycomprise all 6 CDRs from OKT3, as shown below.

Heavy Chain CDR1: (SEQ ID No. 59) KASGYTFTRYTMH CDR2: (SEQ ID No. 60)INPSRGYTNYNQKFKD CDR3: (SEQ ID No. 61) YYDDHYCLDY Light Chain CDR1:(SEQ ID No. 62) SASSSVSYMN CDR2: (SEQ ID No. 63) RWIYDTSKLAS CDR3:(SEQ ID No. 64) QQWSSNPFT

The second binding domain may comprise a scFv which comprises the CDRsequences from OKT3. The second binding domain may comprise the scFvsequence shown below as SEQ IN No. 65 or a variant thereof having atleast 80% sequence identity, which retains the capacity to bind CD3.

SEQ ID No. 65 QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR

A variant sequence from SEQ ID No. 65 may have at least 80, 85, 90, 95,98 or 99% sequence identity and have equivalent or improved CD3 bindingand/or TCR activation capabilities as the sequence shown as SEQ ID No.65.

Bi-Specific T-Cell Engagers (Bites)

BiTES are a new class of therapeutics which approximate a target antigenwith the T-cell receptor (TCR). The original design was of two scFvsconnected together by a linker with one scFv targeting antigen and theother activating a T-cell.

BiTEs are commonly made by fusing an anti-CD3 scFv to an anti-targetantigen scFv via a short five residue peptide linker (GGGGS). In 1995, atandem scFv targeting EpCAM (epithelial 17-1A antigen) and human CD3 inCHO cells was produced. This new kind of bi-specific antibody formatproved to be highly cytotoxic at nanomolar concentrations againstvarious cell lines, using unstimulated human PBMCs in the absence ofco-signaling. Later, a fusion between a murine anti-CD19 scFv and amurine anti-CD3 scFv was created. This molecule demonstrated outstandingin vitro properties, including efficient cytotoxicity, without the needof co-signaling (e.g., through CD28).

Blinatumomab, a murine anti-human CD3×anti-human CD19 was the first BiTEdeveloped and is the most advanced BiTE in clinical trials. Thecandidate is being studied as a treatment of lymphoma and leukemia.

MT110, an anti-human EpCAM x anti-human CD3 TaFv, was the second BiTEtested in clinical trial and the first directed to a wide spectrum ofsolid tumors. In vitro characterizations of MT110 have recapitulated theresults obtained with MT103 on tumor cell lines, thereby demonstratingthe generality of the BiTE format. MT110 is currently in clinical trialfor lung, colorectal and gastrointestinal cancer patients.

The bi-specific molecule of the present invention is based on aBiTE-like format, but instead of having a scFv or other antibody-basedbinding domain binding the target antigen, it has a binding domain basedon the ligand for BCMA, namely APRIL.

This “APRILiTE” format is favourable compared with a classical scFv-scFvformat for various reasons: (a) a single domain—scFv fusion is likelymore stable and easier to make than other formats; (b) the assembly ofBCMA and APRIL on the cell surface require trimerization of each bindingpartner. This induces clustering of T-cell activating domain at aprotein level making the protein highly specific and highly potent.

The molecule of the present invention may comprise one of the followingamino acid sequences, but with a mutation at one of the followingpositions in the portion of the sequence corresponding to APRIL (withreference to the position numbering shown in SEQ ID No. 1): S202, P201,M200, T175, V174, A125, H203, D205 and R206:

SEQ ID No. 66 METDILLLWVLLLWVPGSTGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRSDPAEPKSPDKTHTCPPCPKDPKSGGGGSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRAR AKLNLSPHGTFLGFVKLSEQ ID No. 67 METDTLLLWVLLLWVPGSTGQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSGGGGSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKL SEQ ID No. 68MGTSLLCWMALCLLGADHADGVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKLSGGGSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDSGGGGSQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRS

The molecule of the invention may comprise a variant of the sequenceshown as SEQ ID No. 66, 67 or 68 having at least 80, 85, 90, 95, 98 or99% sequence identity, provided that the variant sequence is a moleculeas defined in the first aspect of the invention, i.e. a bi-specificmolecule which comprises:

-   -   (i) a first domain which binds B cell maturation antigen (BCMA)        and comprises at least part of a proliferation-inducing ligand        (APRIL); and    -   (ii) a second domain capable of activating a T cell.

Signal Peptide

The bi-specific molecule of the invention may comprise a signal peptideto aid in its production. The signal peptide may cause the bi-specificmolecule to be secreted by a host cell, such that the bi-specificmolecule can be harvested from the host cell supernatant.

The core of the signal peptide may contain a long stretch of hydrophobicamino acids that has a tendency to form a single alpha-helix. The signalpeptide may begin with a short positively charged stretch of aminoacids, which helps to enforce proper topology of the polypeptide duringtranslocation. At the end of the signal peptide there is typically astretch of amino acids that is recognized and cleaved by signalpeptidase. Signal peptidase may cleave either during or after completionof translocation to generate a free signal peptide and a mature protein.The free signal peptides are then digested by specific proteases.

The signal peptide may be at the amino terminus of the molecule.

The bi-specific molecule may have the general formula:

Signal peptide—first domain—second domain.

The signal peptide may comprise the SEQ ID No. 69 or 70 or a variantthereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions,substitutions or additions) provided that the signal peptide stillfunctions to cause secretion of the bi-specific molecule.

SEQ ID No. 69: METDTLLLWVLLLWVPGSTG SEQ ID No. 70: MGTSLLCWMALCLLGADHADG

The signal peptides of SEQ ID No. 69 and 70 are compact and highlyefficient. They are predicted to give about 95% cleavage after theterminal glycine, giving efficient removal by signal peptidase.

Spacer

The molecule of the present invention may comprise a spacer sequence toconnect the first domain with the second domain and spatially separatethe two domains.

The spacer sequence may, for example, comprise an IgG1 hinge or a CD8stalk. The linker may alternatively comprise an alternative linkersequence which has similar length and/or domain spacing properties as anIgG1 hinge or a CD8 stalk.

The spacer may be a short spacer, for example a spacer which comprisesless than 100, less than 80, less than 60 or less than 45 amino acids.The spacer may be or comprise an IgG1 hinge or a CD8 stalk or a modifiedversion thereof.

Examples of amino acid sequences for these linkers are given below:

SEQ ID No. 71 (IgG1 hinge): AEPKSPDKTHTCPPCPKDPKSGGGGSSEQ ID No. 72 (CD8 stalk): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

The CD8 stalk has a sequence such that it may induce the formation ofhomodimers (see FIG. 2). If this is not desired, one or more cysteineresidues may be substituted or removed from the CD8 stalk sequence. Thebispecific molecule of the invention may include a spacer whichcomprises or consists of the sequence shown as SEQ ID No. 72 or avariant thereof having at least 80, 85, 90, 95, 98 or 99% sequenceidentity, provided that the variant sequence is a molecule which causesapproximately equivalent spacing of the first and second domains and/orthat the variant sequence causes homodimerisation of the bi-specificmolecule.

The molecule of the invention may have the general formula:

Signal peptide—first domain—spacer—second domain.

The spacer may also comprise one or more linker motifs to introduce achain-break. A chain break separate two distinct domains but allowsorientation in different angles. Such sequences include the sequenceSDP, and the sequence SGGGSDP (SEQ ID No. 73).

The linker may comprise a serine-glycine linker, such as SGGGGS (SEQ IDNo. 74).

Chimeric Antigen Receptors (CARs)

Chimeric antigen receptors (CARs), also known as chimeric T cellreceptors, artificial T cell receptors and chimeric immunoreceptors, areengineered receptors, which graft an arbitrary specificity onto animmune effector cell. In a classical CAR (FIG. 3), the specificity of amonoclonal antibody is grafted on to a T cell or NK cell. CAR-encodingnucleic acids may be introduced into T cells or NK cells using, forexample, retroviral vectors. In this way, a large number ofcancer-specific T cells or NK cells can be generated for adoptive celltransfer. Early clinical studies of this approach have shown efficacy insome cancers, primarily when targeting the pan-B-cell antigen CD19 totreat B-cell malignancies.

The target-antigen binding domain of a CAR is commonly fused via aspacer and transmembrane domain to a signaling endodomain. When the CARbinds the target-antigen, this results in the transmission of anactivating signal to the T-cell it is expressed on.

The CAR may comprise:

(i) a variant APRIL, acting as the B cell maturation antigen(BCMA)-binding domain;(ii) a optional spacer(iii) a transmembrane domain; and(iv) an endodomain.

The endodomain may comprise or associate with an intracellular T-cellsignalling domain.

The CAR of the present invention may comprise one of the following aminoacid sequences, but with a mutation at one of the following positions inthe portion of the sequence corresponding to APRIL (with reference tothe position numbering shown in SEQ ID No. 1): S202, P201, M200, T175,V174, A125, H203, D205 and R206:

SEQ ID No. 75 (dAPRIL-HCH2CH3pvaa-CD28OXZ) METDTLLLWVLLLWVPGSTGSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKLSGGGSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPRSEQ ID No. 76 (dAPRIL-CD8STK-CD28OXZ) METDTLLLWVLLLWVPGSTGSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKLSGGGSDPTTTPAPRPPTPAPTIASQPLSLRPEACIRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID No. 77 (dAPRIL-HNG-CD28OXZ) METDTLLLWVLLLWVPGSTGSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKLSGGGSDPAEPKSPDKTHTCPPCPKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDIQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID No. 78 (dAPRIL-HCH2CH3pvaa-CD28OXZ) MGTSLLCWMALCLLGADHADGKPIPNPLLGLDSTSGGGGSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKLSGGGSDPAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYITLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID No. 79 (dAPRIL-CD8STK-CD28OXZ) MGTSLLCWMALCLLGADHADGKPIPNPLLGLDSTSGGGGSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKLSGGGSDPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSEQ ID No. 80 (dAPRIL-HNG-CD28OXZ) MGTSLLCWMALCLLGADHADGKPIPNPLLGLDSTSGGGGSVLHLVPINATSKDDSDVTEVMWQPALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTFTMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSAGVFHLHQGDILSVIIPRARAKLNLSPHGTFLGFVKLSGGGSDPAEPKSPDKTHTCPPCPKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR

The molecule of the invention may comprise a variant of the sequenceshown as SEQ ID No. 75, 76, 77, 78, 79 or 80 having at least 80, 85, 90,95, 98 or 99% sequence identity, provided that the variant sequence is amolecule as defined in the first aspect of the invention, i.e. a CARwhich comprises:

(i) a BCMA-binding domain;(ii) a optional spacer domain(iii) a transmembrane domain; and(iv) an endodomain;and comprises a mutation at one of the following positions in theportion of the sequence corresponding to APRIL (with reference to theposition numbering shown in SEQ ID No. 1): S202, P201, M200, T175, V174,A125, H203, D205 and R206.

The percentage identity between two polypeptide sequences may be readilydetermined by programs such as BLAST which is freely available athttp://blast.ncbi.nlm.nih.gov.

Nucleic Acid Sequence

The present invention also provides a nucleic acid sequence encoding avariant APRIL, a CAR comprising a variant APRIL or a BiTE comprising avariant APRIL as defined above.

The nucleic acid sequence may be RNA or DNA, it may be double orsingle-stranded.

Nucleic acid sequences encoding APRIL-BiTEs are shown as SEQ ID No.81-83. The nucleic acid sequence of the present invention may encode theamino acid sequence as encoded by SEQ ID No. 81, 82 or 83, but with amutation at one of the following positions in the portion of thesequence corresponding to APRIL (with reference to the positionnumbering shown in SEQ ID No. 1): S202, P201, M200, T175, V174, A125,H203, D205 and R206.

SEQ ID No. 81 (APRILiTE#01)ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCAGCACCGGCCAGGTGCAGCTGCAGCAGAGCGGAGCCGAGCTGGCCAGACCAGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCCGGTACACCATGCACTGGGTGAAGCAGCGGCCAGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCCAGATCGTGCTGACCCAGAGCCCAGCCATCATGAGCGCCAGCCCAGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTGCCAGCCCACTTCAGAGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCGGCATGGAGGCCGAGGATGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCTTCACCTTCGGCAGCGGCACCAAGCTGGAGATCAACCGGTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAAAAGATCCCAAATCTGGCGGAGGCGGCAGCGTGCTGCACCTGGTGCCCATCAACGCCACCAGCAAGGACGACTCTGATGTGACCGAGGTGATGTGGCAGCCAGCCCTGAGACGGGGCAGAGGCCTGCAGGCCCAGGGCTACGGCGTGAGAATCCAGGACGCTGGCGTGTACCTGCTGTACTCCCAGGTGCTGTTCCAGGACGTGACCTTCACAATGGGCCAGGTGGTGAGCCGGGAGGGCCAGGGCAGACAGGAGACCCTGTTCCGGTGCATCCGGAGCATGCCCAGCCACCCCGACAGAGCCTACAACAGCTGCTACAGCGCTGGCGTGTTTCACCTGCACCAGGGCGACATCCTGAGCGTGATCATCCCCAGAGCCAGAGCCAAGCTGAACCTGTCCCCCCACGGCACCTTTCTGGGCTTCGTGAAGCTGTGA SEQ ID No. 82 (APRILiTE#03)ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCAGCACCGGCCAGGTGCAGCTGCAGCAGAGCGGAGCCGAGCTGGCCAGACCAGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCCGGTACACCATGCACTGGGTGAAGCAGCGGCCAGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCCAGATCGTGCTGACCCAGAGCCCAGCCATCATGAGCGCCAGCCCAGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTGCCAGCCCACTTCAGAGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCGGCATGGAGGCCGAGGATGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCTTCACCTTCGGCAGCGGCACCAAGCTGGAGATCAACCGGTCGGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATTCTGGCGGAGGCGGCAGCGTGCTGCACCTGGTGCCCATCAACGCCACCAGCAAGGACGACTCTGATGTGACCGAGGTGATGTGGCAGCCAGCCCTGAGACGGGGCAGAGGCCTGCAGGCCCAGGGCTACGGCGTGAGAATCCAGGACGCTGGCGTGTACCTGCTGTACTCCCAGGTGCTGTTCCAGGACGTGACCTTCACAATGGGCCAGGTGGTGAGCCGGGAGGGCCAGGGCAGACAGGAGACCCTGTTCCGGTGCATCCGGAGCATGCCCAGCCACCCCGACAGAGCCTACAACAGCTGCTACAGCGCTGGCGTGTTTCACCTGCACCAGGGCGACATCCTGAGCGTGATCATCCCCAGAGCCAGAGCCAAGCTGAACCTGTCCCCCCACGGCACCTTTCTGGGCTTCGTGAAGCTG TGASEQ ID No. 83 (APRILiTE#06)ATGGGCACCTCCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGAGCCGACCACGCCGACGGCGTGCTGCACCTGGTGCCCATCAACGCCACCAGCAAGGACGACTCTGATGTGACCGAGGTGATGTGGCAGCCAGCCCTGAGACGGGGCAGAGGCCTGCAGGCCCAGGGCTACGGCGTGAGAATCCAGGACGCTGGCGTGTACCTGCTGTACTCCCAGGTGCTGTTCCAGGACGTGACCTTCACAATGGGCCAGGTGGTGAGCCGGGAGGGCCAGGGCAGACAGGAGACCCTGTTCCGGTGCATCCGGAGCATGCCCAGCCACCCCGACAGAGCCTACAACAGCTGCTACAGCGCTGGCGTGTTTCACCTGCACCAGGGCGACATCCTGAGCGTGATCATCCCCAGAGCCAGAGCCAAGCTGAACCTGTCCCCCCACGGCACCTTTCTGGGCTTCGTGAAGCTGTCTGGAGGCGGCTCGGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATAGCGGTGGCGGTGGCAGCCAGGTGCAGCTGCAGCAGAGCGGAGCCGAGCTGGCCAGACCAGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCCGGTACACCATGCACTGGGTGAAGCAGCGGCCAGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCCCAGCAGAGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGCCGTGTACTACTGCGCCAGATACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGGGCACCACCCTGACCGTGAGCAGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCTCTGGCGGAGGCGGCAGCCAGATCGTGCTGACCCAGAGCCCAGCCATCATGAGCGCCAGCCCAGGCGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTACCAGCAGAAGAGCGGCACCAGCCCCAAGCGGTGGATCTACGACACCAGCAAGCTGGCCAGCGGCGTGCCAGCCCACTTCAGAGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATCAGCGGCATGGAGGCCGAGGATGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACCCCTTCACCTTCGGCAGCGGCACCAAG CTGGAGATCAACCGGTCGTGA

Nucleic acid sequences encoding APRIL-CARs are shown as SEQ ID No.84-89. The nucleic acid sequence of the present invention may encode theamino acid sequence as encoded by SEQ ID No. 84, 85, 86, 87, 88 or 89,but with a mutation at one of the following positions in the portion ofthe sequence corresponding to APRIL (with reference to the positionnumbering shown in SEQ ID No. 1): S202, P201, M200, T175, V174, A125,H203, D205 and R206.

SEQ ID No. 84 (dAPRIL-HCH2CH3pvaa-CD28OXZ)ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCAGCACCGGCAGCGTGCTCCACCTGGTGCCCATCAACGCCACCAGCAAGGACGACTCTGATGTGACCGAGGTGATGTGGCAGCCAGCCCTGAGACGGGGCAGAGGCCTGCAGGCCCAGGGCTACGGCGTGAGAATCCAGGACGCTGGCGTGTACCTGCTGTACTCCCAGGTGCTGTTCCAGGACGTGACCTTCACAATGGGCCAGGTGGTGAGCCGGGAGGGCCAGGGCAGACAGGAGACCCTGTTCCGGTGCATCCGGAGCATGCCCAGCCACCCCGACAGAGCCTACAACAGCTGCTACAGCGCTGGCGTGTTTCACCTGCACCAGGGCGACATCCTGAGCGTGATCATCCCCAGAGCCAGAGCCAAGCTGAACCTGTCCCCCCACGGCACCTTTCTGGGCTTCGTGAAGCTGTCTGGAGGCGGCTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCTAASEQ ID No. 85 (dAPRIL-CD8STK-CD28OXZ)ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCAGCACCGGCAGCGTGCTCCACCTGGTGCCCATCAACGCCACCAGCAAGGACGACTCTGATGTGACCGAGGTGATGTGGCAGCCAGCCCTGAGACGGGGCAGAGGCCTGCAGGCCCAGGGCTACGGCGTGAGAATCCAGGACGCTGGCGTGTACCTGCTGTACTCCCAGGTGCTGTTCCAGGACGTGACCTTCACAATGGGCCAGGTGGTGAGCCGGGAGGGCCAGGGCAGACAGGAGACCCTGTTCCGGTGCATCCGGAGCATGCCCAGCCACCCCGACAGAGCCTACAACAGCTGCTACAGCGCTGGCGTGTTTCACCTGCACCAGGGCGACATCCTGAGCGTGATCATCCCCAGAGCCAGAGCCAAGCTGAACCTGTCCCCCCACGGCACCTTTCTGGGCTTCGTGAAGCTGTCTGGAGGCGGCTCGGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCTAA SEQ ID No. 86 (dAPRIL-HNG-CD28OXZ)ATGGAGACCGACACCCTGCTGCTGTGGGTGCTGCTGCTGTGGGTGCCAGGCAGCACCGGCAGCGTGCTCCACCTGGTGCCCATCAACGCCACCAGCAAGGACGACTCTGATGTGACCGAGGTGATGTGGCAGCCAGCCCTGAGACGGGGCAGAGGCCTGCAGGCCCAGGGCTACGGCGTGAGAATCCAGGACGCTGGCGTGTACCTGCTGTACTCCCAGGTGCTGTTCCAGGACGTGACCTTCACAATGGGCCAGGTGGTGAGCCGGGAGGGCCAGGGCAGACAGGAGACCCTGTTCCGGTGCATCCGGAGCATGCCCAGCCACCCCGACAGAGCCTACAACAGCTGCTACAGCGCTGGCGTGTTTCACCTGCACCAGGGCGACATCCTGAGCGTGATCATCCCCAGAGCCAGAGCCAAGCTGAACCTGTCCCCCCACGGCACCTTTCTGGGCTTCGTGAAGCTGTCTGGAGGCGGCTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCTAASEQ ID No. 87 (dAPRIL-HCH2CH3pvaa-CD28OXZ)ATGGGCACCTCCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGAGCCGACCACGCCGACGGCAAGCCCATTCCCAACCCCCTGCTGGGCCTGGACTCCACCTCTGGCGGAGGCGGCAGCGTGCTGCACCTGGTGCCCATCAACGCCACCAGCAAGGACGACTCTGATGTGACCGAGGTGATGTGGCAGCCAGCCCTGAGACGGGGCAGAGGCCTGCAGGCCCAGGGCTACGGCGTGAGAATCCAGGACGCTGGCGTGTACCTGCTGTACTCCCAGGTGCTGTTCCAGGACGTGACCTTCACAATGGGCCAGGTGGTGAGCCGGGAGGGCCAGGGCAGACAGGAGACCCTGTTCCGGTGCATCCGGAGCATGCCCAGCCACCCCGACAGAGCCTACAACAGCTGCTACAGCGCTGGCGTGTTTCACCTGCACCAGGGCGACATCCTGAGCGTGATCATCCCCAGAGCCAGAGCCAAGCTGAACCTGTCCCCCCACGGCACCTTTCTGGGCTTCGTGAAGCTGTCTGGAGGCGGCTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTCCCGTGGCCGGCCCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCGCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAACCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCT AASEQ ID No. 88 (dAPRIL-CD8STK-CD28OXZ)ATGGGCACCTCCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGAGCCGACCACGCCGACGGCAAGCCCATTCCCAACCCCCTGCTGGGCCTGGACTCCACCTCTGGCGGAGGCGGCAGCGTGCTGCACCTGGTGCCCATCAACGCCACCAGCAAGGACGACTCTGATGTGACCGAGGTGATGTGGCAGCCAGCCCTGAGACGGGGCAGAGGCCTGCAGGCCCAGGGCTACGGCGTGAGAATCCAGGACGCTGGCGTGTACCTGCTGTACTCCCAGGTGCTGTTCCAGGACGTGACCTTCACAATGGGCCAGGTGGTGAGCCGGGAGGGCCAGGGCAGACAGGAGACCCTGTTCCGGTGCATCCGGAGCATGCCCAGCCACCCCGACAGAGCCTACAACAGCTGCTACAGCGCTGGCGTGTTTCACCTGCACCAGGGCGACATCCTGAGCGTGATCATCCCCAGAGCCAGAGCCAAGCTGAACCTGTCCCCCCACGGCACCTTTCTGGGCTTCGTGAAGCTGTCTGGAGGCGGCTCGGATCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCTAA SEQ ID No. 89 (dAPRIL-HNG-CD28OXZ)ATGGGCACCTCCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGAGCCGACCACGCCGACGGCAAGCCCATTCCCAACCCCCTGCTGGGCCTGGACTCCACCTCTGGCGGAGGCGGCAGCGTGCTGCACCTGGTGCCCATCAACGCCACCAGCAAGGACGACTCTGATGTGACCGAGGTGATGTGGCAGCCAGCCCTGAGACGGGGCAGAGGCCTGCAGGCCCAGGGCTACGGCGTGAGAATCCAGGACGCTGGCGTGTACCTGCTGTACTCCCAGGTGCTGTTCCAGGACGTGACCTTCACAATGGGCCAGGTGGTGAGCCGGGAGGGCCAGGGCAGACAGGAGACCCTGTTCCGGTGCATCCGGAGCATGCCCAGCCACCCCGACAGAGCCTACAACAGCTGCTACAGCGCTGGCGTGTTTCACCTGCACCAGGGCGACATCCTGAGCGTGATCATCCCCAGAGCCAGAGCCAAGCTGAACCTGTCCCCCCACGGCACCTTTCTGGGCTTCGTGAAGCTGTCTGGAGGCGGCTCGGATCCCGCCGAGCCCAAATCTCCTGACAAAACTCACACATGCCCACCGTGCCCAAAAGATCCCAAATTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCG CTAA

The nucleic acid sequence may encode the same amino acid sequence asthat encoded by SEQ ID No. 81, 82, 83, 84, 85, 86, 87, 88 or 89comprising the variation mentioned above, but may have a differentnucleic acid sequence, due to the degeneracy of the genetic code. Thenucleic acid sequence may have at least 80, 85, 90, 95, 98 or 99%identity to the sequence shown as SEQ ID No. 81 to 89, provided that itencodes a molecule as defined in the first aspect of the invention.

The nucleic acid sequence may encode the amino acid sequence as encodedby SEQ ID No. 81 to 89, but with one of following the single mutations(SEQ IDs 22 to 45):

-   -   A125T,    -   V174T, V174G,    -   T175H, T175S, T175G,    -   M200C, M200L, M200G, M200S, M200A, M200N,    -   P201V, P201A, P201G, P201R, P201Y, P201W,    -   S202G, S202F, S202D, S202V, S202P, D205P.

The nucleic acid sequence may encode the amino acid sequence as encodedby SEQ ID No. 81 to 89, but with a combination of mutations at thefollowing positions: V174 and T175; or V174 and M200; or V174 and S202;or V175 and M200, or V175 and S202; or D205 and R206; or V174, T175 andM200; or V174, T175 and S202; or T175, D205 and R206; or M200, D205 andR206; or V174, T175, M200 and S202; or T175, S202, D205 and R206;

The nucleic acid sequence may encode the amino acid sequence as encodedby SEQ ID No. 81 to 89, but with one of the following specific mutationcombinations:

-   -   V174T and T175A; or V174T and M200G; or T174S and S202G; or    -   V174T and S202V; or V174G and S202G, or V174G and S202E; or    -   V174G and S202A; or V174G and S202G; or V174E and S202Y; or    -   T175A and S202E; or T175G and S202G; or T175G and S202V; or    -   T175A and S202P; or T175A and M200G; or T175S and S202G; or    -   S202V and H203N; or D205H and R206L; or D205P and R206K; or    -   D205P and R206N; or D205S and R206P; or D205R and R206G; or    -   D205P and R2061; or D205S and R206H; or    -   V174T, T175A and S202E; or V174T, T175A and M200G; or    -   T175A, D205P and R206N; or T175A, D205S and R206H; or    -   M200G, D205P and R206N; or M200G, D205S and R206H; or    -   V174T, T175A, M200G and S202E; or    -   T175A, S202E, D205P and R206N; or    -   T175A, S202E, D205S and R206H.

Vector

The present invention also provides a vector which comprises a nucleicacid sequence according to the present invention. Such a vector may beused to introduce the nucleic acid sequence into a host cell so that itexpresses and produces a variant APRIL according to the first aspect ofthe invention.

The vector may, for example, be a plasmid or synthetic mRNA or a viralvector, such as a retroviral vector or a lentiviral vector.

The vector may be capable of transfecting or transducing an effectorcell.

Cell

The invention also provides a host cell which comprises a nucleic acidaccording to the invention.

The invention also provides a cell which comprises a CAR according tothe invention.

The cell may be an immune cell such as a T-cell or natural killer (NK)cell. It may be a primary cell or a cell from a cell line.

The invention also provides a cell composition comprising a plurality ofCAR-expressing cells of the invention.

The invention also provides a method for making a cell according to thepresent invention which comprises the step of transducing ortransfecting a cell with a vector of the invention which comprises anucleic acid sequence encoding a chimeric antigen receptor.

The cell may be transfected or transduced ex vivo and then reimplantedinto the same or a different subject.

Therapeutic Agent

The present invention provides a therapeutic agent which comprises avariant APRIL, a nucleic acid, a vector a CAR-expressing cell or a BiTEas defined above.

The therapeutic agent may comprise a variant APRIL as the targetingportion, to target the agent to BCMA-expressing cells, such as plasmacells. The therapeutic agent may also comprise a functional domain whichexerts a therapeutic affect, for example by acting directly on theplasma cell or recruiting other cells of the immune system to act on theplasma cell.

The variant APRIL may be conjugated to a drug, such as a cytotoxic drug.

The Variant APRIL may be part of a chimeric antigen receptor, orBispecific T-cell engager (BiTE)

Pharmaceutical Composition

The present invention also relates to a pharmaceutical compositioncontaining a therapeutic agent of the invention together with apharmaceutically acceptable carrier, diluent or excipient, andoptionally one or more further pharmaceutically active polypeptidesand/or compounds. Such a formulation may, for example, be in a formsuitable for intravenous infusion).

Method of Treatment

The therapeutic agent and pharmaceutical composition of the presentinvention may be used for the treatment of a cancerous disease, inparticular a plasma cell disorder or a B cell disorder which correlateswith enhanced BCMA expression.

Plasma cell disorders include plasmacytoma, plasma cell leukemia,multiple myeloma, macroglobulinemia, amyloidosis, Waldenstrom'smacroglobulinemia, solitary bone plasmacytoma, extramedullaryplasmacytoma, osteosclerotic myeloma (POEMS Syndrome) and heavy chaindiseases as well as the clinically unclear monoclonal gammopathy ofundetermined significance/smoldering multiple myeloma.

The disease may be multiple myeloma.

Examples for B cell disorders which correlate with elevated BCMAexpression levels are CLL (chronic lymphocytic leukemia) andnon-Hodgkins lymphoma (NHL). The bispecific binding agents of theinvention may also be used in the therapy of autoimmune diseases likeSystemic Lupus Erythematosus (SLE), multiple sclerosis (MS) andrheumatoid arthritis (RA).

The method of the present invention may be for treating a cancerousdisease, in particular a plasma cell disorder or a B cell disorder whichcorrelates with enhanced BCMA expression.

A method for the treatment of disease relates to the therapeutic use ofan agent or composition of the invention. In this respect, the agent orcomposition may be administered to a subject having an existing diseaseor condition in order to lessen, reduce or improve at least one symptomassociated with the disease and/or to slow down, reduce or block theprogression of the disease. The method of the invention may cause orpromote T-cell mediated killing of BCMA-expressing cells, such as plasmacells.

Diagnosis

The present invention also provides a diagnostic agent for detectingplasma cells which comprises a variant APRIL of the invention.

The diagnostic agent may also comprise a detectable label, such as aradioactive or fluorescent label or a dye.

The diagnostic agent may be for diagnosing a plasma cell disorder.

The diagnostic method may be carried out in vivo or in vitro. In the invivo method, the diagnostic agent is administered to the subject.

In the in vitro method, the variant APRIL is added to a sample from thesubject in vitro. The sample may comprise plasma cells. The sample maybe or be derived from a blood sample, such as a PBMC sample.

The invention will now be further described by way of Examples, whichare meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention.

Examples Example 1—Characterisation of BCMA as a Target for Myeloma

Primary myeloma cells were isolated by performing a CD138 immunomagneticselection on fresh bone marrow samples from Multiple myeloma patientsthat were known to have frank disease. These cells were stained with theBCMA specific J6MO mAb (GSK) which was conjugated to PE. At the sametime, a standard of beads with known numbers of binding sites wasgenerated using the PE Quantibrite bead kit (Becton Dickenson) as perthe manufacturer's instructions. The BCMA copy number on myeloma cellscould be derived by correlating the mean-fluorescent intensity from themyeloma cells with the standard curve derived from the beads. It wasfound that the range of BCMA copy number on a myeloma cell surface islow: at 348.7-4268.4 BCMA copies per cell with a mean of 1181 and amedian of 1084.9 (FIG. 2). This is considerably lower than e.g. CD19 andGD2, classic targets for CARs. Presence of BCMA expression on primarymyeloma cells was also confirmed with the Vicky-1 antibody (AbcamAb17323), examples of which are shown in FIG. 18.

Example 2—Design and Construction of APRIL Based CARs

APRIL in its natural form is a secreted type II protein. The use ofAPRIL as a BCMA binding domain for a CAR requires conversion of thistype II secreted protein to a type I membrane bound protein and for thisprotein to be stable and to retain binding to BCMA in this form. Togenerate candidate molecules, the extreme amino-terminus of APRIL wasdeleted to remove binding to proteoglycans. Next, a signal peptide wasadded to direct the nascent protein to the endoplasmic reticulum andhence the cell surface. Also, because the nature of spacer used canalter the function of a CAR, three different spacer domains were tested:an APRIL based CAR was generated comprising (i) a human IgG1 spaceraltered to remove Fc binding motifs; (ii) a CD8 stalk; and (iii) theIgG1 hinge alone (cartoon in FIG. 4 and amino acid sequences in FIGS.5A-5C, and also amino acid sequences in FIGS. 19A-19C which differ fromthe sequences in FIG. 5 by having a different signal peptide and the V5epitope tag). These CARs were expressed in a bicistronic retroviralvector (FIG. 6A) so that a marker protein—truncated CD34 could beco-expressed as a convenient marker gene.

Example 3—Expression and Function of APRIL Based CARs

The aim of this study was to test whether the APRIL based CARs which hadbeen constructed were expressed on the cell surface and whether APRILhad folded to form the native protein. T-cells were transduced withthese different CAR constructs and stained using a commerciallyavailable anti-APRIL mAb, along with staining for the marker gene andanalysed by flow-cytometry. The results of this experiment are shown inFIG. 6B where APRIL binding is plotting against marker genefluorescence. These data show that in this format, the APRIL based CARsare expressed on the cell surface and APRIL folds sufficiently to berecognized by an anti-APRIL mAb.

Next, it was determined whether APRIL in this format could recognizeBCMA and TACI. Recombinant BCMA and TACI were generated as fusions withmouse IgG2a-Fc. These recombinant proteins were incubated with thetransduced T-cells. After this, the cells were washed and stained withan anti-mouse fluorophore conjugated antibody and an antibody to detectthe marker gene conjugated to a different fluorophore. The cells wereanalysed by flow cytometry and the results are presented in FIG. 6C. Thedifferent CARs were able to bind both BCMA and TACI. Surprisingly, theCARs were better able to bind BCMA than TACI. Also, surprisingly CARswith a CD8 stalk or IgG1 hinge spacer were better able to bind BCMA andTACI than CAR with an Fc spacer.

Example 4—APRIL Based Chimeric Antigen Receptors are Active Against BCMAExpressing Cells

T-cells from normal donors were transduced with the different APRIL CARsand tested against SupT1 cells either wild-type, or engineered toexpress BCMA and TACI. Several different assays were used to determinefunction. A classical chromium release assay was performed. Here, thetarget cells (the SupT1 cells) were labelled with ⁵¹Cr and mixed witheffectors (the transduced T-cells) at different ratio. Lysis of targetcells was determined by counting ⁵¹Cr in the co-culture supernatant(FIG. 6A shows the cumulative data, example data from a single assaywith different effector:target ratios is shown in FIG. 16).

In addition, supernatant from T-cells cultured 1:1 with SupT1 cells wasassayed by ELISA for Interferon-gamma (FIG. 6B shows cumulative data,example data from a single assay is shown in FIG. 17). Measurement ofT-cell expansion after one week of co-culture with SupT1 cells was alsoperformed (FIG. 6C). T-cells were counted by flow-cytometry calibratedwith counting beads. These experimental data show that APRIL based CARscan kill BCMA expressing targets. Further, these data show that CARsbased on the CD8 stalk or IgG1 hinge performed better than the Fc-pvaabased CAR.

Example 5—APRIL Based CARs are Able to Kill Primary Myeloma Cells

The above data are encouraging since they demonstrate that it inprinciple, it is possible to make an APRIL based CAR. However, sincemost primary myeloma cells express a low number of BCMA molecules ontheir surface, it was investiagated whether such an APRIL based CARwould cause killing of primary myeloma cells, particularly in cases withlow-density expression. Three cases were selected which represented therange of BCMA expression described in FIG. 2: the first had dimexpression (lower than mean); the second case had intermediateexpression (approximately mean expression) and the third had bright(above mean expression). FIG. 8 shows a histogram of BCMA stainingagainst isotype control for all three cases on the left to illustrateBCMA expression. Since when comparing APRIL based CARs with differentspacers it had been determined that CARs with CD8 stalk spacer and IgG1hinge spacer performed better than the Fc-pvaa spacered CAR, in thisassay, only the CD8 stalk and hinge APRIL CARs were tested. On the left,survival of myeloma cells compared with starting numbers is shown at day3 and day 6 after a 1:1 co-culture of myeloma cells and CAR T-cells. Byday 6, >95% of the myeloma cells were eliminated, including those withdim BCMA expression. Dim BCMA expressing myeloma cells can be targetedby the APRIL CARs albeit with a slower tempo of killing than higherexpressers.

Example 6—Construction of a Series of “APRILITES”

The present inventors have constructed a series of bi-specific engagerswhich connect a scFv from OKT3 to the extracellular domain of APRIL, asshown in FIG. 24A. Several design considerations were made during theconstruction of these molecules: (a) the proteoglycan binding aminoterminus of APRIL was truncated to prevent non-specific binding; (b) inconstructs 4, 5 and 6, a signal peptide was attached to the matureectodomain of APRIL; (c) the OKT3 was re-formatted as a scFv with alinker connecting the heavy and light chain variable regions; (d)various different spacers were tried between the scFv and APRIL.

The various different formats were as follows:

(1) OKT3 scFv connected to truncated APRIL by the IgG1 hinge;(2) OKT3 scFv connected to truncated APRIL via a (SGGGGS)3 linker;(3) OKT3 scFv connected to truncated APRIL via the CD8 stalk;(4) truncated APRIL connected to OKT3 scFv via an IgG1 hinge;(5) truncated APRIL connected to the OKT3 scFv via a (SGGGGS)3 linker;and(6) truncated APRIL connected to the OKT3 scFv via a CD8 spacer.

Constructs (3) and (6) form homodimers through disulphide bonds in theCD8 spacer. The amino acid sequences for constructs (1), (3) and (6) areshown in FIGS. 30A-30C.

Example 7—Expression of APRILiTEs in 293T Cells

293 T cells were transfected with expression plasmids coding for theAPRILiTE constructs listed above. Supernatant from the 293T cells wasrun on an acrylamide gel and proteins transferred to a membrane. Themembrane was then stained with an antibody which recognized APRIL. Theresults are shown in FIG. 25. Proteins 1, 3 and 6 were detected at theexpected molecular weight. Proteins 2, 4 and 5 were not detected,indicating that these configurations are unstable.

Example 8—Binding to TCR and BCMA

It was then investigated whether these proteins could bind either theT-cell receptor (TCR) on one end, and BCMA on the other end. Supernatantfrom 293T cells transfected was used to stain Jurkat T-cells and aJurkat T-cell clone which has TCRαβ knocked out. This demonstrates theAPRILiTE binds the TCR (FIG. 26b ). SupT1 cells engineered to expressBCMA and SupT1 cells engineered to express TACI were then stained withthe above supernatant, using a secondary anti-APRIL biotin followed bystreptavidin PE. The results are shown in FIG. 26a . It was found thatAPRILiTES 1,3 and 6 bound BCMA, and TACI to a lesser extent.

Example 9—Stable APRILITEs Trigger IFNγ Release

Normal donor T-cells were cultivated 1:1 with different SupT1s. TheSupT1s used were either non-transduced, engineered to express BCMA orengineered to express TACI. The results are shown in FIG. 27. It wasfound that T-cells only released IFNγ in the presence of either APRILiTEwhen exposed with SupT1-cells engineered with BCMA or TACI. The responseto BCMA was greater than that with TACI.

Example 10—Stable APRILITEs Trigger T-Cell Mediated Killing of BCMA+Targets

T-cells were cultured 1:1 with wild-type SupT1 cells, SupT1 cellsexpressing BCMA and SupT1 cells expressing TACI in the absence of or inthe presence of APRILiTEs 1,3 and 6. The results are shown in FIG. 28.The remaining T-cells are shown as a proportion of SupT1 cells presentin the condition with no APRILiTE added.

Example 11—Investigating BCMA Expression on Primary Myeloma Cells

Four different myeloma samples were stained with the rat anti-human BCMAmAb Vicky1. The results are shown in FIG. 29. In clinically andmorphologically typical myelomas (panels 2 to 4) intermediate or dimstaining is seen.

Example 12—Investigating the Effect of APRILiTEs on Primary MyelomaCells

Left over material from a diagnostic bone-marrow aspirate from twopatients with known multiple BCMA+ myeloma was used. A CD138 magneticbead selection was performed to purify myeloma cells from the aspirate.These cells were rested in complete culture medium for 48 hours andstaining for BCMA was performed to check that they were in fact BCMApositive. It was found that the myeloma cells express BCMA but at lowlevels (FIG. 31).

Next, normal donor peripheral mononuclear cells which had beenstimulated using OKT3 and CD28.2 were CD56 depleted to remove NK cells.A 1:1 co-culture of CD56 depleted PBMCs and CD138 selected primaryMyeloma cells were performed in the absence or presence of eitherAPRILITE#03 and #06. Insufficient material was present to testAPRILiTE#01. The co-cultures were observed by microscopy. Interferongamma release into supernatant was measured by ELISA. Survival ofmyeloma cells was measured by Annexin V/PI staining and bead-countcontrolled flow-cytometry.

Clear clumping (a sign of T-cell activation) was seen upon co-culture(see FIG. 32). Interferon-gamma release was observed in conditions wherePBMCs were cultured with Myeloma cells in the presence of the APRILiTES,albeit at less absolute amounts than when co-cultured with SupT1. BCMAcells (FIG. 33). Killing of Myeloma cells was also observed when PBMCswere present with APRILiTE after 6 days of co-culture (FIG. 34).

These findings demonstrate that APRILiTEs cause T cell activation in thepresence of primary myeloma cells at a level sufficient to cause T-cellmediated killing of the myeloma cells.

Example 13—Testing the APRILiTES In Vivo

A huSCID model is used: NSG (nod-scid gamma, NOD-scid IL2Rgamma^(null))mice are xenografted with a myeloma cell line which expresses typicallevels of BCMA. These lines are engineered to express firefly Luciferaseto measure disease by bioluminescence imaging. Normal donor PBMCs areadministered via the tail vein during concomitant intraperitonealadministration of APRILiTEs. The following are sequentially measured (1)serum levels of APRILiTEs; (2) serum levels of human Interferon-gamma;(3) peripheral blood T-cell expansion, engraftment and activation byflow cytometry; (4) Bioluminescence measurement of tumour. At take-down,the following are measured: (1) tumour burden by marrow histology; (2)T-cell proliferation and engraftment by flow cytometry of marrow,spleen, blood and lymph nodes; and (3) the remaining tissues areexamined grossly and immunohistochemically for any toxicity.

Example 14—Production of APRIL Mutants Particularly Suited to TargetingBCMA

The aim was to generate APRIL mutants whose binding may be more suitablefor

CAR. Using crystallographic data described by Hymowitz et al, 2004, TheJournal of biological chemistry: Volume 280; Issue 8; Pages 7218-27 andfrom RCSB deposits 1XU1 and 1XU2, several residues were selected whichmay alter binding to BCMA or may increase specificity to BCMA over TACI.

A strategy to identify mutations at these residues with usefulproperties is outlined in FIG. 31B. Using splicing by overlap PCR witholigonucleotides degenerate over codons for mutation, libraries ofmutant APRILs were generated randomized over key mutants. Theselibraries were ligated into a scaffold shown in FIG. 31A which presentsAPRIL on a CD8 stalk and co-expresses CD34 with a foot-and-mouth 2Apeptide. Typical expression from this construct is shown in FIGS. 9A-9C.These ligation products were transformed into competent bacteria, singlecolonies picked, individually expanded and the DNA was extracted andtransfected into 293T cells.

The 293T cells were subsequently incubated separately with eitherrecombinant human BCMA-Fc or TACI-Fc. Cells were then washed andsecondarily stained with Jackson polyclonal anti-Fc Alexa fluor 488 andthe marker gene stained with anti-CD34 APC. The APRIL mutants werescreened in this manner in batches with wild-type APRIL and a CD34 onlyas controls in each batch. CD34+ve events were split into 4 gatesnumbered as shown in FIG. 31. The Alexa fluor 488 median fluorescenceindex (MFI) was calculated for each gate and average gradient betweenMFI of various gates was calculated using the formula:[(MFI.1-MFI.2)+(MFI.2-MFI.3)+(MFI.3-MFI.4)]/3 (illustrated in FIG. 31C).

In this way, an average MFI gradient was calculated for binding to BCMAand TACI for each APRIL mutant. For each mutant, the average MFIgradient of BCMA and TACI binding was converted to a ratio of binding toAPRIL WT control in each batch. Plasmids giving rise to potentiallyuseful mutants were sequenced by capillary sequencing.

The results of this initial screening are summarized in Table 1 andillustrated in FIG. 10.

Classes of mutants were then combined together by a similar strategy tothat outlined for single mutants, but mutant APRIL coding plasmid wasused as template to introduce further mutations. The results of thiswork are summarized in Table 2 and illustrated in FIG. 11. It waspossible to generate mutants with much higher affinity to BCMA thanwild-type: for instance mutant D205R, R206G; we were able to generatemutants with BCMA binding equal to wild-type APRIL but no binding toTACI—for instance mutant T175A, S202P. We were also able to generatemutants with lower binding to BCMA than wild-type (which mayparadoxically improve recognition of low-density antigen), but nobinding of TACI—for instance mutant V174T, T175A, M200G, S202E.

Larger scale, higher quality plasmid DNA from the most promising mutantswas generated and repeat transfection and expression data was performed.These data are shown in FIGS. 12A-12F.

Example 15—Secreted and Truncated APRIL Fused to an Fc Spacer RecognizesBCMA and TACI

In order to investigate whether truncated APRIL in a CAR format (i.e.fused to a transmembrane domain and anchored to a cell membrane) couldbind BCMA and TACI, a basic CAR was engineered in frame with theself-cleaving foot and mouth disease 2A peptide with truncated CD34, asa convenient marker gene. A stable SUPT1 cell line was established whichexpresses this construct. Secreted truncated BCMA and TACI fused tohuman (and other species, not shown) Ig Fc domain was also generated andrecombinant protein produced. It was shown that both BCMA-Fc and TACI-Fcbind the engineered SUPT1 cell line. Only cells expressing the CD34marker gene were found to bind BCMA-Fc and TACI-Fc (FIGS. 9A-9C).

Example 16—APRIL Based Chimeric Antigen Receptors are Stably Expressedon the Surface of T-Cells

The CAR spacer domain can alter sensitivity and specificity. Threeversions of an APRIL-based CAR were generated with three spacer domains:(i) a human IgG1 spacer altered to remove Fc binding motifs; (ii) a CD8stalk; and (iii) the IgG1 hinge alone (FIG. 14B). Primary human T-cellswere transduced with these different CARs and stained using acommercially available anti-APRIL mAb (FIG. 15).

Example 17—APRIL Based Chimeric Antigen Receptors are Active AgainstCognate Target Expressing Cells

T-cells from normal donors were transduced with the different APRIL CARsand tested against SupT1 cells either wild-type, or engineered toexpress BCMA and TACI. Several different assays were used to determinefunction. A classical chromium release assay was performed. Here, thetarget cells (the SupT1 cells) were labelled with ⁵¹Cr and mixed witheffectors (the transduced T-cells) at different ratio. Lysis of targetcells was determined by counting ⁵¹Cr in the co-culture supernatant(FIG. 16).

In addition, supernatant from T-cells cultured 1:1 with SupT1 cells wasassayed by ELISA for Interferon-gamma (FIG. 17).

Measurement of T-cell expansion after one week of co-culture with SupT1cells was also performed. T-cells were counted by flow-cytometrycalibrated with counting beads. Initial data (not shown) appears toindicate that the CD8 stalk based construct results in more T-cellproliferation than the other constructs.

Example 18—Production of BCMA-Specific APRIL Mutants

APRIL mutants were generated using degenerate primers targeting specificcodons. The codons were identified through in silico analysis ofAPRIL-BCMA and APRIL-TACI binding. From this analysis, residues thatseemed involved in TACI binding but not BCMA binding were targeted.

Plasmids were produced encoding (i) cell surface expressed CD34 and (ii)the APRIL mutants. The plasmids were then transformed into bacteria,plated, single colonies picked, individually expanded and the DNA wasextracted and transfected into 293T cells.

T cells expressing a single APRIL mutant and CD34 were each aliquotedinto two and incubated separately with 0.1 μg RND human BCMA-hFc orTACI-hFc chimera. Cells were then washed and secondarily stained withJackson polyclonal ahFc Alexa fluor 488 and BD aCD34 APC.

The APRIL mutants were screened in this manner in batches with wild-typeAPRIL as a control in each batch. CD34+ve events were split into 4 gatesnumbered as shown in FIGS. 9A-9C. The Alexa fluor 488 medianfluorescence index (MFI) was calculated for each gate and averagegradient between MFI of various gates was calculated using the formula:[(MFI.1-MFI.2)+(MFI.2-MFI.3)+(MFI.3-MFI.4)]/3.

In this way, an average MFI gradient was calculated for binding to BCMAand TACI for each APRIL mutant. For each mutant, the average MFIgradient of BCMA and TACI binding was converted to a ratio of binding toAPRIL WT control in the relevant screened batch. The mutants whichshowed a higher BCMA:TACI binding ratio than wild type were thensequenced.

The results are shown in FIGS. 20A-20W and the sequences of key mutantsare shown in FIG. 21A-21B.

The effect of glycine substitution was then examined at the targetedresidues. The results, which are shown in FIG. 22, show that residuesS202, P201, M200, T175, V174, A125, H203, D205 and R206 on APRIL_(wt)are comparatively more important for binding to TACI than BCMA.

Example 19—Demonstration of In Vivo Function of APRIL CAR T-Cells

In order to demonstrate APRIL CAR T-cell function in vivo, APRIL CART-cells were tested in a human/mouse chimeric model.

MM1.s (ATCC CRL-2974) is a human myeloma cell line which expressesintermediate levels of BCMA. The inventors engineered this cell line toexpress firefly Luciferase to derive the cell-line MM1.s.FLuc.

NOD scid gamma (NSG: NOD.Cg-Prkdc^(scid) II2rgtm1^(WjI/SzJ)) mice areprofoundly immunosuppressed mice capable of engrafting several humancell lines and human peripheral blood lymphocytes. Three month oldfemale NSG mice received 1×10⁷ MM1.s.FLuc cells vial tail-vein injectionwithout any preparative therapy. Engraftment was determined by serialbioluminescence imaging (FIGS. 23A-23B). Robust and increasingintramedullary engraftment was observed in all mice. At day 13, 5×10⁶APRIL-HNG-CD28OXZ CAR T-cells were administered via tail vein injection.Serial bioluminescence was performed which showed rapid decrease inburden of MM1.s (FIGS. 23A-23B) in all treated mice to a completeremission. This response to CAR therapy was confirmed by flow-cytometryand immunohistochemistry.

Example 20—Testing Function of Various APRIL Mutants in a BiTE Format

Four normal donor PBMCs were incubated with SupT1 cells, SupT1 cellsengineered to express BCMA, SupT1 cells engineered to express TACI oralone in the presence of different BiTES based on either WT APRIL orvarious mutants. Interferon-gamma levels were measured 24 hours later.The results are shown in FIG. 35. The mutant M200G shows significantlyimproved BCMA vs TACI specificity than wild-type.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, cellular immunology or related fields are intended tobe within the scope of the following claims.

1-19. (canceled)
 20. A therapeutic agent which comprises a variantproliferation-inducing ligand (APRIL) conjugated to a drug.
 21. Thetherapeutic agent according to claim 20 wherein the drug is a cytotoxicdrug.
 22. The therapeutic agent according to claim 20, wherein thevariant APRIL comprises the BCMA-binding site of the sequence shown asSEQ ID No.
 1. 23. The therapeutic agent according to claim 20, whereinthe variant APRIL comprises a truncated APRIL.
 24. The therapeutic agentaccording to claim 23, wherein the truncated APRIL retains BCMA and TACIbinding but lacks the capacity to bind proteoglycan.
 25. The therapeuticagent according to claim 23, wherein the truncated APRIL lack the aminoterminal 116 amino acids from the sequence shown shown as SEQ ID No.
 126. The therapeutic agent according to claim 23, wherein the truncatedAPRIL comprises the sequence shown as SEQ ID No. 2
 27. The therapeuticagent according to claim 20, wherein the variant APRIL has a higherbinding affinity to BCMA than wild-type APRIL; and/or altered bindingkinetics compared with wild-type APRIL, and/or a higher BCMA:TACI(transmembrane activator and calcium modulator and cyclophilin ligandinteractor) binding ratio than wild-type APRIL and which comprisesmutations at one or more of the following positions: A125, V174, T175,M200, P201, S202, H203, D205 and R206.
 29. A pharmaceutical compositionwhich comprises a therapeutic agent according to claim 20 and apharmaceutically acceptable carrier, diluent or excipient.
 30. A methodfor treating a plasma cell disorder which comprises the step ofadministering the pharmaceutical composition according to claim 29 to asubject.
 31. The method according to claim 30, wherein the plasma celldisorder is selected from plasmacytoma, plasma cell leukemia, multiplemyeloma, macroglobulinemia, amyloidosis, Waldenstrom'smacroglobulinemia, solitary bone plasmacytoma, extramedullaryplasmacytoma, osteosclerotic myeloma, heavy chain diseases, monoclonalgammopathy of undetermined significance and smoldering multiple myeloma.32. The method according to claim 31, wherein the plasma cell disorderis multiple myeloma.